The microenvironment, or niche, in which hematopoietic stem cells (HSC) reside, is essential for their regulation. Since HSC number limits their clinical use, strategies to increase HSC through niche manipulation could increase the scope of their therapeutic application. The long-term objective of this proposal is to manipulate the bone marrow microenvironment to increase HSC numbers and thereby their clinical utility. To meet this objective, we established a novel experimental model in which treatment with parathyroid hormone (PTH) and/or activation of its receptor (PTH1R) in osteoblastic cells expands Long-term HSC (LT-HSC), thus identifying osteoblastic cells as key regulators of HSC. While defining mediators of PTH action in the niche, we discovered that Prostaglandin E2 (PGE2), which is released by osteoblastic cells upon PTH treatment, selectively expands Short-term HSC (ST-HSC) in vivo. Such exquisite and specialized regulation of HSC subsets has not previously been achieved, but we believe that it will have important therapeutic implications. Therefore, we have developed two unique experimental tools to define HSC regulation. Based on our observations, we hypothesize that independent mechanisms selectively regulate LT-HSC and ST-HSC and that these have different effects on HSC regulation. To demonstrate this hypothesis, this proposal will pursue three specific aims. In Aim1, the specific cellular and molecular mechanisms that mediate LT-HSC expansion in response to PTH will be defined in vivo by pharmacologic and genetic means. In Aim2, the specific cellular and molecular mechanisms that mediate PGE2-dependent ST-HSC expansion will be defined in vivo and in vitro. In Aim 3, interactions between regulation of LT-HSC and ST-HSC will be established. Data from the studies proposed in this application will 1) define the PTH and PGE2-activated mechanisms regulating LT-HSC and ST-HSC; 2) identify interactions between these mechanisms which could be exploited for targeted regulation of HSC subsets in the setting of specific therapeutic need. The studies proposed are therefore designed to result in findings that not only advance the understanding of stem cell regulation, but also devise pharmacologic strategies that can be brought back to patients, directly impacting their clinical care. The availability of PTH and PGE2 receptor specific agonists makes these studies particularly timely for future translation to human therapy. Using two novel in vivo experimental tools, this project will determine specific regulatory mechanisms, direct and indirect, that control HSC behavior, and that can stimulate differentially subsets of HSC which have different properties. Since HSC give rise to all blood cells, these regulatory mechanisms could be therapeutically exploited to increase HSC in specific situations of blood cell injury or deficiency.